This invention relates in general to a system for detection of sample components and in particular to a system for detection of sample components where the difference in sensitivity to detection of the components moving at different velocities is compensated for.
Historically, most electrophoretic and chromatographic separation techniques have employed off-line detection systems. Detection in high performance liquid chromatography is typically performed in a post-column flow cell and in slab gel electrophoresis, measurement of bands is often accomplished by staining the gel after electrophoretic separation. Recently, a variety of separation techniques have taken advantage of on-line detection to simplify analysis and improve reliability. As described in L. M. Smith, et al., Nature, 321:674-679 (1986), Smith et al. developed a fluorescence system for on-line detection with a polyacrylamide tube gel that measured DNA bands as they migrated past the focus of a laser beam. This procedure eliminated the need for manual analysis and the requirement for running multiple, overlapping gels. A similar approach that used 1 fluorescence detection for slab gel electrophoresis has been described by Middendorf et al. See, for example, L. R. Middendorf, et al., Electrophoresis, 13:487-494 (1992). Microcolumn procedures capable of separating low volume samples have employed on-line detection almost exclusively because of the ease of implementation -- the separation channel is often made of fused silica and is therefore optically transparent -- and the relative difficulty of making off-column plumbing connections with low-volume channels.
In addition to fluorescence detection, other detection methods have also been used in electrophoretic and chromatographic separation techniques, such as absorbance, electrochemical, chemiluminescence, radioisotope and four-wave mixing. In most of the above-mentioned detection methods, including fluorescence detection, the situation often arises where the detection sensitivity is limited by shot noise, which follows Poissons statistics. In such cases, it is useful for all analytes to be detected in an unbiased manner so that the detection signal can be faithfully related to the concentration of the analyte band in the detection zone. This goal is not achieved in traditional on-line detection techniques because of difficulties which may be inherent in the detection process.
In fluorescence detection, for example, quantitation often has involved labeling the analytes of interest with the same fluorescent tag and treating the fluorescent signal as proportional only to the fluorescence quantum yield and absorption cross-section of the tag and the concentration of the analyte. Frequently, a fluorescently-tagged standard having a known concentration is added to the sample mixture. A laser is then directed to the migrating sample components as they pass a detection zone to excite the components into light emission. The laser, however, also photobleaches portions of the components, thereby rendering such portions permanently undetectable. The detection process can destroy the detectable qualities in a number of other detection techniques other than fluorescence detection, such as in absorbance, electrochemical, chemiluminescence and four-wave mixing detection. In on-line detection systems, different components pass the zone at different velocities, and a greater fraction of the slower moving components may be photobleached than the faster components so that slower components exhibit smaller peaks if the detection is optimized for the faster components. In conventional on-line detection techniques, such effects are ignored, and the size of the corresponding peak is referenced to determine the concentration of a known species. Such approach does not take into account the effects of band velocities on detection sensitivity or the fact that the process of detection itself may destroy the detectability of fractions of the analyte band.
From the above, the traditional detection approach not only causes inaccuracies in quantitation, but may fail to detect a component altogether. Where the amount of a sample component is small so that it is barely detectable at optimum detection conditions, such component will become undetectable if detection conditions are optimized for a component different from such component as would be the case in traditional on-line detection. It is therefore desirable to provide an improved detection system for use in sample separation in which the above-described difficulties are alleviated.